A new integrated method for tissue extracellular vesicle enrichment and proteome profiling

Abstract

Extracellular vesicles (EVs) are membranous vesicles released by cells that carry a number of biologically important components such as lipids, proteins, and mRNAs. EVs can mediate cancer cell migration, invasion, angiogenesis, and cell survival, greatly contributing to cell-to-cell communication in the tumor microenvironment. Additionally, EVs have been found to have diagnostic and prognostic significance in various cancers. However, the direct isolation of pure EVs remains challenging, especially from tissue samples. Currently available EV isolation approaches, e.g., ultracentrifugation, are time-consuming, instrumental dependent, and have a low recovery rate with limited purity. It is urgent to develop rapid and efficient methods for enriching tissue EVs for biological and clinical studies. Here, we developed a novel isolation approach for tissue EVs using an extraction kit combined with TiO₂ microspheres (kit-TiO₂). The EVs were first precipitated from the tissue fluid using a precipitation agent and then further enriched using microspheres based on the specific interaction between TiO₂ and the phosphate groups on the lipid bilayer of the EVs. Kit-TiO₂ approach led to improved purity and enrichment efficiency of the isolated EVs, as demonstrated by western blot and proteomic analysis, compared with previously reported methods. A total of 1966 protein groups were identified from the tissue EVs. We compared the proteomic profiles of the liver tissue EVs from healthy and hepatocellular carcinoma (HCC) bearing-mice. Twenty-five significantly upregulated and 75 downregulated protein groups were found in the HCC EVs. Among the differentially expressed proteins, Atic, Copa, Cont3, Me1, Anxa3, Fth1, Anxa5, Phb1, Acaa2, ATPD, and Glud1 were reported to be highly relevant to HCC. This novel isolation strategy has provided a powerful tool for enriching EVs directly from tissues, and may be applied in biomarker discovery and drug screening of HCC.